We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on differentiation/dedifferentiation pathways and protection against oxidative or inflammatory stress. The role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In addition, given the likely importance of noncoding RNAs (including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs)) as regulators of gene expression in the response of RPE cells to various signals, we are interested in determining how changes in miRNAs and lncRNAs expression in RPE cells affect differentiation/dedifferentiation, and how their expression may be changed by agents/factors with which RPE cells are treated in our experiments. In the past year we have made progress in the following areas: 1) We continued a project studying the role of noncoding RNAs in inflammatory processes in RPE. Both miRNAs and lncRNAs are involved in many aspects of regulation of gene transcription, post-transcriptional regulation (splicing, etc.), epigenetic regulation, and such programmatic functions as X-inactivation. Changes in expression of certain lncRNAs are associated with diseases such as various cancers and with Alzheimers disease. It is not unreasonable to expect that there will also be changes in lncRNAs associated with eye diseases such as AMD. Using our RNAseq datasets we have identified lncRNAs that show changes (both up- and down-regulation) in our ARPE-19 differentiation model. Manipulation of expression of these lncRNAs results in changes to expression of a variety of important protein-coding genes. In connection with this, we found that a combination of IFNgamma, IL1beta and TNFalpha increased the expression of BANCR in ARPE-19 cells. BANCR is a lncRNA recently shown to regulate EMT and metastasis. IFNgamma by itself increased the expression of BANCR while the other two cytokines did not show any noticeable effect. The increase in BANCR expression by IFNgamma involved activation of STAT1. JAK Inhibitor I, which blocks JAK/STAT, effectively suppressed both the induction of BANCR and STAT1 activation. Thus, BANCR could potentially act as a link between RPE dysfunction and inflammatory response and, therefore, may play a role in the pathology of AMD. A manuscript describing these results was published in this reporting period. 2) We continued a project focusing on lncRNA discovery in RPE, to identify and characterize lncRNAs important in RPE gene regulation. Non-coding RNAs (miRNAs and lncRNAs) play an important role in regulating the expression of genes involved in numerous biological processes including cellular differentiation. Most ncRNA studies have focused on microRNAs (miRNAs/miRs), as we have previously studied in RPE, while few studies have investigated how lncRNAs, a group of RNAs (>200 nucleotides, generally with no protein coding potential) regulate gene expression and cellular differentiation in RPE. To regulate RPE-specific functions we expect there to be a set of highly RPE-specific lncRNAs. Also, there is likely to be an important role of lncRNAs in the differentiation of RPE cells. As native human RPE is difficult to obtain, we are using bovine RPE to develop the paradigm, working with native and RPE primary cultures to identify lncRNAs differentially expressed between RPE and retina, and between native bovine and cultured RPE cells. These studies are ongoing. 3) We continued a project to study the role of the lncRNA LINC00276. RNASeq analysis provided a comprehensive view of differentially expressed lncRNAs in differentiated 4-month old ARPE-19 cells relative to 4-day old cells. From these data, we observed a number of lncRNAs that were differentially regulated with fold change of 2.5 or greater in differentiated ARPE-19 cells and which show a differential expression pattern between 4-day and 4-month cultured cells. The expression of one of these in particular, LINC00276, was increased >200-fold in 4-month cells compared to 4-day cells. Knockdown of LINC00276 negatively affected expression of various RPE-preferential transcripts, while its overexpression enhanced expression. Currently, we are trying to determine interaction partners (e.g., protein, RNA) of LINC00276. These studies are ongoing. 4) We continued a project to examine the expression of secreted proteins (secretome) in differentiated ARPE-19 cultured in DMEM with pyruvate for 4 months and exhibiting native-like RPE phenotype. We are also investigating secretion of exosomes from ARPE-19 as a function of their differentiation to determine proteins and/or RNAs secreted. We continue to collaborate with sections in the LRCMB and with other laboratories and sections (Molecular Structure and Functional Genomics, Laboratory of Immunology), as well as with extramural labs in the analysis (HPLC and mass spectrometry) of retinoid, lipids, and other compounds.